Structural, Catalytic, Binding and Immunological Properties Associated with each of the two subunits of rat liver ligandin: rat liver ligandin consists of two subunits of 22,000 (A) and 25,000 (B) daltons, which were purified to homogeneity. Amino acid analysis of A and B and limited proteolytic digest reveal that the two subunits are closely related but structurally distinct. Most of the cysteine residues of ligandin are on B and linkage of A B dimer to Thiol-Sepharose is mediated by B. A was purified by Thiol-Sepharose affinity and QAE-A50 Sephadex chromatography. A homodimers had glutathione-S-transferase specific activities similar to those of native ligandin. Circular dichroism spectra showed that A A has two high affinity bilirubin binding sites as opposed to 1:1 stoichiometric rations of the natural A B heterodimer. In the heterodimer, A preferentially bound 35S-sulfobromophthalein covalently after incubation and ultra-violet irradiation. When the binding site was blocked by covalent modification, the protein no longer absorbed on BSP-sepharose affinity columns. Purified ligandin-sulfobromophthalein complexes were separated from unreacted ligandin by affinity chromatography and contained both subunits, retained immunological reactivity, but lost high affinity binding capacity. Although glutathione-S-transferase activity is associated with each subunit of ligandin, the high affinity binding capacity of the protein is associated with A. Structural, catalytic, binding and immunological properties associated with each of the two subunits of rat liver ligandin: rat testicular and liver cystols contain ligandin as determined immunologically, and have high glutathione-S-transferase activity. Unlike liver cytosol, testicular cytosol does not contain protein components that bind bilirubin or sulfobromophthalein with high affinity. To investigate these effects, ligandin was purified to homogeneity from rat testis. Whereas rat liver ligandin consists of equal amounts of 2 subunits with molecular weights of 22,000 (A) and 25,000 (B), more than 90% of testicular ligandin consists of B. Rat testicular ligandin is immunologically similar to liver ligandin, and has identical glutathione-S-transferase activity, but lacks the capacity for high affinity binding of bilirubin and sulfobromophthalein. The amino acid composition and other properties of testicular ligandin (Text Truncated - Exceeds Capacity)